1. Field of the Invention
The invention relates to genes and genetic suppressor elements associated with the control of neoplastic transformation of mammalian cells. More particularly, the invention relates to methods for identifying such genes and genetic suppressor elements as well as to uses for such genes and genetic suppressor elements. The invention specifically provides genetic suppressor elements derived from genes associated with the transformed phenotype of mammalian cells, and therapeutic and diagnostic uses related thereto. The invention also provides genes associated with the control of neoplastic transformation of mammalian cells.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United States. Clinically, a broad variety of medical approaches, including surgery, radiation therapy and chemotherapeutic drug therapy are currently being used in the treatment of human cancer (see the textbook CANCER: Principles & Practice of Oncology, 2d Edition, De Vita et al., eds., J. B. Lippincott Company, Philadelphia, Pa., 1985). However, it is recognized that such approaches continue to be limited by a fundamental lack of a clear understanding of the precise cellular bases of malignant transformation and neoplastic growth.
The beginnings of such an understanding of the cellular basis of malignant transformation and neoplastic growth have been elucidated over the last ten years. Growth of normal cells is now now understood to be regulated by a balance of growth-promoting and growth-inhibiting genes, known as proto-oncogens and tumor suppressor genes, respectively. Proto-oncogene are turned into oncogenes by regulatory or structural mutations that increase their ability to stimulate uncontrolled cell growth. These mutations are therefore manifested as dominant (e.g. mutant RAS genes) or co-dominant (as in the case of amplification of oncogenes such as N-MYC or HER2/NEU) (see Varmus, 1989, "A historical overview of oncogenes", in Oncogenes and the Molecular Origin of Cancer, Weinberg, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 3-44).
Dominant and co-dominant genes can be effectively identified and studied using many different techniques based on gene transfer or on selective isolation of amplified or overexpressed DNA sequences (Kinzler et al., 1987, Science 236: 70-73; Schwab et al., 1989, Oncogene 4: 139-144; Nakatani et al., Jpn. J. Cancer Res. 8: 707-710). Expression selection has been successfully used to clone a number of cellular oncogenes. The dominant nature of the oncogenes has facilitated the analysis of their function both in vitro, in cell culture, and in vivo, in transgenic animals. Close to fifty cellular oncogenes have been identified so far (Hunter, 1991, Cell 64: 249-270).
It is likely, however, that there are at least as many cancer-associated genes that are involved in suppression rather than induction of abnormal cell growth. This class of genes, known as anti-oncogenes or tumor suppressors, has been defined as comprising "genetic elements whose loss or inactivation allows a cell to display one or another phenotype of neoplastic growth deregulation" by Weinberg (1991, Science 254: 1138-1146). Changes in a tumor suppressor gene that result in the loss of its function or expression are recessive, because they have no phenotypic consequences in the presence of the normal allele of the same gene. The recessive nature of mutations associated with tumor suppressors makes such genes very difficult to analyze or identify by gene transfer techniques and explains why oncogene research is far more advanced than studies of tumor suppressors.
In normal cells, tumor suppressor genes may participate in growth inhibition at different levels, from the recognition of a growth inhibiting signal and its transmission to the nucleus, to the induction (or inhibition) of secondary response genes that finally determine the cellular response to the signal. The known tumor suppressor genes have indeed been associated with different steps of the regulatory pathway. Thus, the DCC and ErbA genes encode receptors of two different classes (Fearon et at., 1990, Science 242: 49-56; Sap et al., 1986, Nature 324: 635-640; Weinberger et al., 1986, Nature 324: 641-646). The gene NF-1 encodes a polypeptide that resembles ras-interacting proteins, that are members of the signaling pathway (Xu et al., 1990, Cell 62: 599-608; Ballester et al., 1990, cell 62: 851-859; Buchberg et al., 1990, Nature 347: 291-294; Barbacid, 1987, Ann. Rev. Biochem. 56: 779-827). p53, RB and WT genes encode nuclear regulatory proteins (Fields et al., 1990, Science 249: 1046-1049; Raycroft et al., 1990, Science 249: 1049-1051; Kern et al., 1991, Oncogene 6: 131-136; O'Rourke et al., 1990, Oncogene 5: 1829-1832; Kern et al., 1991, Science 252: 1708-1711; Lee et al., 1987, Nature 329: 642-645; Friend et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9059-9063; Call et al., 1990, Cell 60: 509-520; Gessler et al., 1990, Nature 343: 774-778).
Two approaches have been previously used for cloning tumor suppressor genes. The first approach is based on isolating the regions associated with nonrandom genetic deletions or rearrangements observed in certain types of tumors. This approach requires the use of extremely laborious linkage analyses and does not give any direct information concerning the function of the putative suppressor gene (Friend et al., 1991, Science 251: 1366-1370; Viskochil et al., 1990, Cell 62: 187-192; Vogelstein et al., 1988, N. Engl. J. Med. 319: 525-532). In fact, among numerous observations of loss of heterozygosity in certain tumors (Solomon et al., 1991, Science 254: 1153-1160; LaForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88: 5036-5040; Trent et al., 1989, Cancer Res. 49: 420-423), there are only a few examples where the function of the affected gene is understood. In two of these rare cases the gene function was identified using another method, analysis of dominant negative mutant proteins (Herskowitz, 1987, Nature 329: 219-222).
Specifically, the tumor suppressor genes erbA and p53 were first discovered as altered forms which encoded mutant proteins (Sap et al., 1986, ibid.; Weinberger et al., 1986, ibid.; Raycroft et al., 1990, ibid.; Milner et al., 1991, Molec. Cell. Biol. 11: 12-19). These altered genes were initially classified as oncogenes, since they induced cell transformation when transfected alone or in combination with other oncogenes (ras in the case of p53 and erbB in the case of erbA; see Eliyahu et al., 1984, Nature 312: 646-649; Parada et al., 1984, Nature 312: 649-651; Graf & Beug, 1983, Cell 34: 7-9; Damm et al., 1989, Nature 339: 593-597). Later, however, it was recognized that both of these "oncogenes" acted by interfering with the normal function of the corresponding wild-type genes. Thus, the oncogenic mutant p53 protein forms functionally inactive complexes with the wild-type protein; such complexes fail to provide the normal negative regulatory function of the p53 protein (Herskowitz, 1986, ibid.; Milner et al., 1991, ibid.; Montenarh & Quaiser, 1989, Oncogene 4: 379-382; Finlay et al., 1988, Molec. Cell. Biol. 8: 531-539). The oncogene erbA, found in chicken erythroblastosis virus, is a mutant version of the chicken gene for thyroid hormone receptor, the transcriptional regulatory protein which participates in the induction of erythroid differentiation (Damm et al., 1989, ibid.; Damm et al., 1987, EMBO J. 6: 375-382). The mutant ErbA protein blocks the function of the wild-type receptor by occupying its specific binding sites in the DNA (Sap et al., 1989, Nature 340: 242-244).
Thus, naturally arising dominant negative mutants not only allowed the identification of the corresponding tumor suppressor genes but also served as tools for their functional analysis. Such natural tools for recessive gene identification seem to be rare, however, limiting the utility of this approach for the discovery of new tumor suppressor genes.
The discovery and analysis of new recessive genes involved in neoplastic transformation may be greatly accelerated through the use of genetic suppressor elements (GSEs), derived from such genes and capable of selectively suppressing their function. GSEs are dominant negative factors that confer the recessive-type phenotype for the gene to which the particular GSE corresponds. Recently, some developments have been made in the difficult area of isolating recessive genes using GSE technology. Roninson et al., U.S. Pat. No. 5,217,889 (issued Jun. 8, 1993) teach a generalized method for obtaining GSEs (see also Holzmayer et al., 1992, Nucleic Acids Res. 20: 711-717). Gudkov et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 3231-3235 teach isolation of GSEs from topoisomerase II cDNA that induce resistance to topoisomerase II-interactive drugs. Co-pending U.S. patent applications Ser. No. 08/033,986, filed Mar. 9, 1993, and Ser. No. 08/177,571, filed Jan. 5, 1994, disclosed the discovery by the present inventors of the novel and unexpected result that GSEs isolated from RNA of cells resistant to the anticancer DNA damaging agent, etoposide, include a GSE encoding an antisense RNA homologous to a portion of a kinesin heavy chain gene. Additionally, co-pending U.S. patent application Ser. No. 08/033,986 disclosed two other GSEs from previously-unknown genes, the expression of said GSEs conferring etoposide resistance on mammalian cells. Co-pending U.S. patent application Ser. No. 08/199,900, filed Feb. 22, 1994, disclosed GSEs from previously-unknown genes, the expression of said GSEs conferring cisplatin resistance on mammalian cells.
These results further underscored the power of the GSE technology developed by these inventors to elucidate recessive gene-mediated biological phenomenon involving unexpected mechanisms, including drug resistance in cancer cells, thereby providing the opportunity and the means for overcoming drug resistance in cancer patients. This technology has now been applied to isolating and identifying GSEs that confer the transformed phenotype of malignant mammalian cells in previously untransformed cells expressing such GSES, and for isolating and identifying genes associated with the transformed phenotype.